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N-terminally green fluorescent protein (GFP)-tagged regulator of G protein signaling (RGS) 2 and RGS4 fusion proteins expressed in human embryonic kidney 293 cells localized to the nucleus and cytosol, respectively. They were selectively recruited to the plasma membrane by G proteins and correspondingly by receptors that activate those G proteins: GFP-RGS2 when coexpressed with G␣s, ␤ 2 -adrenergic receptor, G␣q, or AT 1A angiotensin II receptor, and GFP-RGS4 when coexpressed with G␣i2 or M 2 muscarinic receptor. G protein mutants with reduced RGS affinity did not produce this effect, implying that the recruitment involves direct binding to G proteins and is independent of downstream signaling events. Neither agonists nor inverse agonists altered receptor-promoted RGS association with the plasma membrane, and expressing either constitutively activated or poorly activated G protein mutants produced effects similar to those of their wild-type counterparts. Thus, intracellular interactions between these proteins seem to be relatively stable and insensitive to the activation state of the G protein, in contrast to the transient increases in RGS-G protein association known to be caused by G protein activation in solution-based assays. G protein effects on RGS localization were mirrored by RGS effects on G protein function. RGS4 was more potent than RGS2 in promoting steady-state Gi GTPase activity, whereas RGS2 inhibited Gsdependent increases in intracellular cAMP, suggesting that G protein signaling in cells is regulated by the selective recruitment of RGS proteins to the plasma membrane.

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Regulator of G protein signaling (RGS) proteins limitG protein-coupled receptors (GPCRs) 6 respond to a variety of hormones, paracrine factors, and neurotransmitters by activating heterotrimeric G proteins. Upon activation of the G protein, G␣ is stimulated to exchange bound GDP for cytosolic GTP and is thought to subsequently dissociate from the ␤␥ dimer. Both G␣ and the ␤␥ dimer are then capable of interacting with cellular effectors for a period of time that is limited by the intrinsic GTPase activity of the G␣ subunit. Regulator of G protein signaling (RGS) proteins can reduce the duration of these interactions by increasing the rate of GTP hydrolysis by the G␣ subunits, or by otherwise blocking interactions between G␣ and its target enzymes through a poorly understood process sometimes referred to as "effector antagonism" (1, 2).G protein signaling in osteoblasts is a critical regulator of bone formation. The predominant GPCRs expressed by osteoblasts include the parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor type 1 (PTH1R), P2Y nucleotide receptors, and prostaglandin receptors (3). Other GPCRs found in osteoblasts include endothelin, adenosine, ␤-adrenergic, angiotensin II, and calcium-sensing receptors (3, 4). Binding of PTH to its specific receptor, PTH1R, predominantly leads to the activation of G s (although under certain conditions the receptor can also couple to G q and G i ) (5, 6). Activated G s stimulates adenylyl cyclase to generate intracellular cAMP, which results in the activation of protein kinase A. Stimulation of G q promotes the activity of phospholipase C-␤ (PLC-␤), resulting in the accumulation of inositol 1,4,5-trisphosphate and diacylglycerol, which lead, respectively, to release of calcium from intracellular stores and activation of protein kinase C. Nucleotide stimulation of P2Y receptors in osteoblasts results in PLC-␤ activation and calcium mobilization, with no effect on adenylyl cyclase (3, 7).